Inhibiting subthalamic D5 receptor constitutive activity alleviates abnormal electrical activity and reverses motor impairment in a rat model of Parkinson's disease

Abstract

Burst firing has been reported as a pathological activity of subthalamic nucleus (STN) neurons in Parkinson's disease. However, the origin of bursts and their causal link with motor deficits remain unknown. Here we tested the hypothesis that dopamine D5 receptors (D5Rs), characterized by a high constitutive activity, may contribute to the emergence of burst firing in STN. We tested whether inhibiting D5R constitutive activity depresses burst firing and alleviates motor impairments in the 6-OHDA rat model of Parkinson's disease. Intrasubthalamic microinjections of either an inverse agonist of D5Rs, flupenthixol, or a D2R antagonist, raclopride, were applied. Behavioral experiments, in vivo and in vitro electrophysiological recordings, and ex vivo functional neuroanatomy studies were performed. Using [(5)S]GTPγ binding autoradiography, we show that application of flupenthixol inhibits D5R constitutive activity within the STN. Furthermore, flupenthixol reduced evoked burst in brain slices and converted pathological burst firing into physiological tonic, single-spike firing in 6-OHDA rats in vivo. This later action was mimicked by calciseptine, a Cav1 channel blocker. Moreover, the same treatment dramatically attenuated motor impairment in this model and normalized metabolic hyperactivity in both STN and substantia nigra pars reticulata, the main output structure of basal ganglia in rats. In contrast, raclopride as well as saline did not reverse burst firing and motor deficits, confirming the selective action of flupenthixol on D5Rs. These results are the first to demonstrate that subthalamic D5Rs are involved in the pathophysiology of Parkinson's disease and that administering an inverse agonist of these receptors may lessen motor symptoms.

Figure 1.

Flupenthixol weakens evoked bursts in burst-competent neurons and minimizes spontaneous burst firing in vitro. Aa, Representative examples of the action of flupentixol, butaclamol, and fluphenazine on bursts (top traces) evoked by depolarizing stimuli (bottom traces) in DA-intact animals. The time course of the action of flupenthixol on the duration of evoked burst (black squares), the number (gray diamonds), and the frequency (white squares) of action potentials (AP) is shown below the traces. Inverted triangles point to the three examples. Ab, Box-plot summary changes in typical features of evoked bursts after application of the three drugs. Ac, Flupenthixol turned burst firing into irregular firing in a spontaneously burst-firing neuron. B, Flupenthixol reduced evoked bursts in neurons from DA-depleted animals. Note that the bath contained fast synaptic transmission inhibitors and raclopride, a D₂R inhibitor. Wilcoxon's matched-pairs signed-rank test, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

Constitutive activity of D₅Rs in the rat STN blocked by flupenthixol. A–C, Histograms showing the dose–response effects of the D₁/D₅ receptor agonist SKF 38393 (A) and antagonist SCH 23390 (B), as well as the D₅R inverse agonist flupenthixol (C) on [³⁵S]GTPγ binding in the STN, assessed by OD. D, SCH 23390 blocked the effects of both SKF 38393 and flupenthixol. Data represent mean ± SEM of OD values. One-way ANOVA (A–C) and unpaired t test (D). *p < 0.05, **p < 0.01, compared with basal OD.

Figure 3.

Flupenthixol reverses pathological burst-firing activity of STN neurons. A, A representative example of spike trains recorded in the STN in a sham-depleted rat. B, A representative photomicrograph of the striatum after immunohistochemistry of tyrosine hydroxylase in a brain slice from a DA-depleted rat. Note the extensive lesion of fibers in the striatum ipsilateral to the side that received stereotactic 6-OHDA injection into the medial forebrain bundle. C, Representative examples of spike trains recorded in the STN in DA-depleted rats before (left) and after local injection of flupenthixol (Flu, right). D, E, Histograms (D) and diagrams (E) of the effects of flupenthixol on STN neurons discharging in tonic single spike or burst mode. F, Histograms of burst frequency, percentage of spikes in burst, and firing rate of STN neurons before and after flupenthixol injection into the STN. G, Histograms of burst frequency, percentage of spikes in burst, and firing rate of STN neurons before and after local injection of calciseptine. Note that calciseptine in the same way as flupenthixol turned the pathological burst-firing activity into tonic, single-spike firing. Data represent mean ± SEM. D, χ² test, **p < 0.01 compared with sham-depleted rats, ^$$p < 0.05 compared with DA-depleted rats. F, G, Mann–Whitney test, *p < 0.05, **p < 0.01; ns, not significant.

Figure 4.

Raclopride does not induce any effect on the pathological burst-firing activity of STN neurons. A, Representative examples of spike trains recorded in the STN in DA-depleted rats before (left) and after local injection of raclopride (Raclo, right) into the STN. B, C, Histograms (B) and diagrams (C) of the effects of raclopride on STN neurons discharging in tonic single-spike or burst mode. Note that raclopride did not reverse the pathological burst-firing activity into tonic, single-spike firing. D, Histograms of burst frequency, percentage of spikes in burst, and firing rate of STN neurons before and after raclopride injection into the STN. Data represent mean ± SEM. B, χ² test, **p < 0.01 compared with sham-depleted rats. D, Mann–Whitney test, *p < 0.05; ns, not significant.

Figure 5.

Flupenthixol attenuates motor impairment in DA-depleted rats. A, Locomotor activity in sham-lesioned rats (n = 10, white bars) and 6-OHDA unilaterally lesioned rats was measured before (black bars) and after (gray bars) local injection of flupenthixol (Flu, n = 19) or raclopride (Raclo, n = 15) into the STN (4 μg/200 nl). Only flupenthixol improved locomotor activity in 6-OHDA-lesioned rats. B, Asymmetry in paw use was partially but significantly reversed by local injection of flupenthixol into the STN (n = 10). Values are presented as the mean ± SEM. *p < 0.05, ***p < 0.0001; ns, not significant. Full and dashed lines, Mann–Whitney and Wilcoxon's matched-pairs signed-rank tests, respectively. Ipsi, Ipsilateral; Cont, contralateral.

Figure 6.

Flupenthixol normalizes the metabolic hyperactivity in the STN and SNr. A, C, Representative photomicrographs of the metabolic activity revealed by COx staining in the STN (A) and SNr (C). B, D, STN (B) and SNr (D) hyperactivity in DA-depleted rats (black bars) compared with sham-lesioned animals (white bars) was normalized by injecting flupenthixol (Flu) into the STN (gray bar). Data represent mean ± SEM of OD values. **p < 0.001, ***p < 0.0001; ns, not significant. Scale bar, 250 μm. Note that, in addition to the increased COx levels in the STN and SNr, this figure shows an increased COx level in the zona incerta (ZI), as described previously in hemiparkinsonian rats (Périer et al., 2000). ic, Internal capsule.